Electronic Supplementary Information

EJNMMI Radiopharmacy and Chemistry

Convergent Synthesis of 13N-labelled Peptidic Structures Using Aqueous [13N]NH3

Julia E. Blower, Samuel F. Cousin and Antony D. Gee

Division of Imaging Sciences and School of Biomedical Engineering and Imaging Sciences, King’s College London, UK. SE1 7EH; Fax: 020 718 85442; Tel: 020 718 88366; Email:

Contents

General Materials and Methods

General Procedure for Preparation of Reference Compounds

General Procedure for Preparation of Radiolabelled Compounds

Characterisation Data for Reference Compounds

Ugi versus Passerini structures

References

General Materials and Methods

Benzaldehyde (≥99%), levulinic acid (98%), p-toluic acid (98%), tert-butyl isocyanide (98%) andtrifluoroacetic acid (99%) were purchased from Sigma-Aldrich. Benzyl isocyanide (98%) and 2,2,2-trifluoroethanol (99.8%) were purchased from Acros Organics. 1,1,3,3-tetramethylbutyl isocyanide and ammonia solution (28-30%) were purchased from Merck KGaA. Acetic acid (~100%) was purchased from AnalaR. Methanol (HPLC grade) was purchased from Fisher.

Reactions were carried out using a CEM Discover microwave synthesis unit.

1H-NMR and 13C-NMR spectra were obtained using a Bruker DRX 400 MHz spectrometer. Mass spectroscopy was performed on an Agilent Technologies 6520 Accurate-Mass Q-TOF LC/MS connected to an Agilent Technologies1200 HPLC system with UV detector and autosampler.

Radio-HPLC analysis was performed on an Agilent Technologies 1200 Series with UV detector (254 nm) and Lablogic β+ radio-detector using an Agilent Eclipse XDB-C18 column (5 μm, 4.6 × 150 mm). The following mobile phase conditions were used: solvent A: H2O + 0.1% TFA; solvent B: MeOH + 0.1% TFA; time:%B 0:5, 1:5, 10:95, 18:5, 23:5. Flow rate was 1 mL/min

General Procedure for Preparation of Reference Compounds

Method was based on procedure reported by Thompsonet al[1].

Entries 1-5

Benzaldehyde (2 mmol), carboxylic acid (2 mmol), isocyanide (2 mmol) and ammonia (excess, ~ 3mmol)were combined in 2,2,2-trifluoroethanol (2 mL) in a microwave tube. The mixture was heated at 100 oC for 30 min in a microwave synthesis unit with stirring. The reaction mixture was cooled to room temperature and the solvent was removed under vacuum using a rotary evaporator. The crude product was then filtered and washed with ice cold methanol. The desired product was then isolated using semi-preparative HPLC and lyophilised.

Entries 6-7

Levulinic acid (2 mmol), isocyanide (2 mmol) and ammonia (excess, ~ 3mmol)were combined in 2,2,2-trifluoroethanol (2 mL) in a microwave tube. The mixture was heated at 100 oC for 30 min in a microwave synthesis unit with stirring. The reaction mixture was cooled to room temperature and the solvent was removed under vacuum using a rotary evaporator. The crude product was then filtered and washed with ice cold methanol. The desired product was then isolated using semi-preparative HPLC and lyophilised.

Semi-preparative HPLC was performed on an Agilent Technologies 1200 Series with UV detector (254 nm) using an Agilent XDB-C18 column (5 μm, 9.4 × 250 mm). The following mobile phase conditions were used: solvent A: H2O + 0.1% TFA; solvent B: MeOH + 0.1% TFA; time:%B 0:0, 1:0, 55:100, 75:100. Flow rate was 3 mL/ min.

General Procedure for Preparation of Radiolabelled Compounds

[13N]NH3 production

Aqueous [13N]NH3 was produced on a CTI RDS 112 biomedical cyclotron via the 16O(p,α)13N nuclear reaction. The target contained 8 mL H2O with 5 mM ethanol and was irradiated with 11.2MeV protons at a beam current of 30 μA for 20 min. The irradiated solution was pumped from the cyclotron through narrow bore PEEK tubing to the radiochemistry laboratory where it is passed through an IC-OH cartridge (Maxi-Clean™, Grace Davison Discovery Sciences) conditioned with water (5 mL), to remove impurities.

Concentration of [13N]NH3 into a smaller volume (1 mL) was carried out by loading the solution on to a weak cation exchange Sep-Pak (Accell Plus CM Light, Waters.) conditioned with water (5 mL), and eluted with saline (0.9%, 1 mL).

Entries 1-5

Benzaldehyde (48.5 μmol), carboxylic acid (48.5 µmol), isocyanide (48.5 μmol), ammonium hydroxide solution (28-30%, 10 µL, 148 µmol) and [13N]NH3 (50 μL) were combined in 2,2,2-trifluoroethanol (200 μL) in a microwave tube. The mixture was heated at 120 oC for 10 min in a microwave synthesis unit with stirring. The reaction mixture was cooled to room temperature and analysed via radio-HPLC. Isolation of 1 was carried out using semi-preparative radio-HPLC.

Semi-preparative radio-HPLC was performed on an Agilent Technologies 1200 Series with UV detector (254 nm) and Lablogic β+ radio-detector using an Agilent Eclipse XDB-C18 column (5 µm, 9.4 × 250 mm). The following mobile phase conditions were used: solvent A: H2O + 0.1% TFA; solvent B: MeOH + 0.1% TFA; time (min):%B 0:50, 1:50, 5:95, 10:95, 15:5, 18:5. Flow rate was 3 mL/min.

Entries 6-7

Levulinic acid (48.5 µmol), isocyanide (48.5 μmol), ammonium hydroxide solution (28-30%, 10 µL, 148 µmol) and [13N]NH3 (50 μL) were combined in 2,2,2-trifluoroethanol (200 μL) in a microwave tube. The mixture was heated at 120 oC for 10 min in a microwave synthesis unit with stirring. The reaction mixture was cooled to room temperature and analysed via radio-HPLC.

Preparation of 1 using conventional heating methods

Benzaldehyde (48.5 μmol), carboxylic acid (48.5 µmol), isocyanide (48.5 μmol), ammonium hydroxide solution (28-30%, 5 µL, 74 µmol) and [13N]NH3 (50 μL) were combined in 2,2,2-trifluoroethanol (200 μL) in a sealed reaction vessel. The mixture was heated at 100 oC for 15 min. The reaction mixture was cooled to room temperature and analysed via radio-HPLC. The RCY was 8% (c.f. microwave heating: Table 1, entry 1, RCY = 13%).

Characterisation Data for Reference Compounds

2-acetamido-2-phenyl-N-(2,4,4-trimethylpentan-2-yl)acetamide (1);white solid;1H NMR (CDCl3, 400 MHz): δ 7.31-7.20 (m, 5H), 6.94 (bd,J= 6.7 Hz, 1H), 5.66 (bs, 1H), 5.30 (d, J= 6.8 Hz, 1H), 1.94 (s, 3H), 1.55 (q, J=14.9 Hz,2H), 1.27 (s, 6H), 0.76 (s, 9H);13C NMR (CDCl3, 400 MHz): δ 169.6, 168.6, 138.4, 128.9, 128.7, 128.2, 55.9, 52.1, 31.2, 31.4, 29.02, 28.4, 23.2.

LC/MS (ESI+): calculated for C18H29N2O2 [M+H]+: 305.2224; found 305.2225.

4-methyl-N-(2-oxo-1-phenyl-2-((2,4,4-trimethylpentan-2-yl)amino)ethyl)benzamide (2);white solid; 1H NMR (CDCl3, 400 MHz): δ 7.71 (d, J= 8.2 Hz, 2H), 7.68 (bs, 1H), 7.43-7.18 (m, 7H), 5.72 (bs, 1H), 5.50 (d, J= 6.3 Hz ,1H), 2.35 (s, 3H), 1.60 (q, J= 15 Hz, 2H), 1.32 (s, 6H), 0.79 (s, 9H);13C NMR (CDCl3, 400 MHz): δ 168.66, 166.44, 142.12, 138.66, 131.10, 129.16, 129.00, 128.22, 127.36, 127.19, 57.91, 55.86, 52.16,31.45, 31.26, 29.08, 28.50, 21.48

LC/MS (ESI+): calculated for C24H33N2O2 [M+H]+: 381.2537; found 381.2549.

2-acetamido-N-benzyl-2-phenylacetamide (3);white solid; 1H NMR (CDCl3, 400 MHz): δ 7.42-7.13 (m, 10H), 7.03 (bd, J=6.7 Hz, 1H), 6.57 (bs, 1H), 5.62 (d, J=7.1 Hz,1H), 4.41 (qd, J= 16.0, 5.5 Hz, 2H),1.97 (s, 3H);13C NMR (CDCl3, 400 MHz): δ 170.03, 169.73, 138.07, 137.48, 129.03, 128.68, 128.41, 127.55, 127.53, 127.29, 57.02, 43.76, 23.13.

LC/MS (ESI+): calculated for C17H19O2N2 [M+H]+: 283.1441; found 283.1461.

N-(2-(tert-butylamino)-2-oxo-1-phenylethyl)-4-methylbenzamide (4);white solid; 1H NMR (CDCl3, 400 MHz): δ 7.75 (d,J=7.9 Hz,2H), 7.66 (bs, 1H), 7.48 (d, J=7.4 Hz, 2H), 7.38-7.22 (m, 5H), 5.84 (bs, 1H), 5.63 (d, J=6.5 Hz, 1H), 2.41 (s, 3H), 1.32 (s, 9H);13C NMR (CDCl3, 400 MHz): δ 169.22, 166.50, 142.17, 138.80, 131.04, 129.17, 128.99, 128.19, 127.26, 127.20, 57.47, 51.90, 28.57, 21.48.

LC/MS (ESI+): calculated for C20H25N2O2[M+H]+: 325.1911; found 325.1941.

2-acetamido-N-(tert-butyl)-2-phenylacetamide (5);white solid; 1H NMR (CDCl3, 400 MHz): δ 7.40-7.31 (m, 5H), 6.88 (bd, J=6.2 Hz, 1H), 5.51 (bs, 1H), 5.38 (d, J=7.1 Hz, 1H), 2.03 (s, 3H), 1.31 (s,9H);13C NMR (CDCl3, 400 MHz): δ 169.48, 169.02, 138.57, 129.03, 128.29, 127.27, 57.16, 51.91, 28.54, 23.27.

LC/MS (ESI+): calculated for C14H21N2O2 [M+H]+: 249.1598; found 249.1618.

N-benzyl-2-methyl-5-oxopyrrolidine-2-carboxamide (6);colourless oil;1H NMR (CDCl3, 400 MHz): δ 7.38-7.28 (m, 5H), 6.75 (bs, 1H), 6.68 (bs, 1H), 4.46 (s, 2H), 2.55-2.49 (m, 1H), 2.42 (t,J=7.8 Hz, 2H), 2.15-2.07(m, 1H), 1.58 (s, 3H);13C NMR (CDCl3, 400 MHz): δ 178.91, 173.80, 137.65, 128.86, 127.82, 127.81, 63.54, 43.90, 33.66, 29.97, 25.42.

LC/MS (ESI+): calculated for C13H17O2N2 [M+H]+: 233.1285; found 233.1286.

N-(tert-butyl)-2-methyl-5-oxopyrrolidine-2-carboxamide (7);colourless oil; 1H NMR (CDCl3, 400 MHz): δ 7.21 (bs, 1H), 6.12 (bs, 1H), 2.78 (m, 1H), 2.64 (m, 1H), 2.48 (m, 1H), 2.08 (m, 1H), 1.53 (s, 3H), 1.37 (s, 9H);13C NMR (CDCl3, 400 MHz): δ 176.95, 173.17, 64.16, 51.69, 37.81, 30.10, 28.50, 25.29.

LC/MS (ESI+): calculated for C10H19O2N2 [M+H]+: 199.1441; found 199.1456.

Ugi versus Passerini structures

General structure of a Ugi reaction product (A) versus a Passerini reaction product (B).

References

  1. M. J. Thompson and B. Chen, J. Org. Chem., 2009, 74, 7084-7093.

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